The present invention relates to methods of presenting polymeric biomolecules such as nucleic acids for analysis, and in particular, to methods for tagging and elongating the biomolecules for characterization, sorting and analysis or other use.
A single DNA molecule can be elongated, for example, by shear forces of liquid flow, by capillary action or by convective flow, and then fixed in an elongated form to a substrate by electrostatic attraction. Sequence features of the fixed molecule can be identified by cleaving the fixed molecule with one or more restriction enzymes to produce gaps that can be marked by fluoroscopic markers or the like and then visualized. See, e.g., U.S. Pat. Nos. 6,713,263; 6,610,256; 6,607,888; 6,509,158; 6,448,012; 6,340,567; 6,294,136; 6,221,592. See also, U.S. Published Patent Application Nos. 2005/0082204; 2003/0124611; 2003/0036067. Each patent and published patent application is incorporated herein by reference as if set forth in its entirety. Although fixing the DNA to the substrate simplifies the analysis by preserving the elongated state, stabilizing the position of the molecule, and preventing fragment shuffling after cleavage, it is difficult to control chemical interactions with the DNA because of its close proximity to the substrate. Also, cleaved DNA may not be not suited for subsequent use, especially where fragment shuffling diminishes information content that can be obtained from an uncleaved molecule.
One can avoid such surface effects by suspending the DNA molecule in a “nanochannel” without attachment to the channel walls. A nanochannel is a channel having a cross-sectional dimension less than 1,000 nanometers and typically on the order of 30 nanometers. The suspended DNA molecule is sufficiently spaced apart from the channel walls to avoid surface interference in the reaction process while still stabilizing the DNA molecule sufficiently for analytical techniques.
Practical use of nanochannels faces a number of obstacles. Nanochannels are extremely difficult to fabricate, thus costly, and as a practical matter, are not reusable. In addition, the small size of nanochannels makes the addition of chemical reagents, especially enzymes, difficult. It is also difficult to encourage DNA molecules to enter the small cross-sectional area of nanochannels. Likewise, it can be difficult to remove interfering reaction by-products from nanochannels. Finally, because DNA is not under significant tension within nanochannels, restriction enzymes cutting the DNA may not make visible gaps.